The present invention relates to a process for formation and anion-exchange of crystalline salts of an echinocandin nucleus, in particular, salts of an Echinocandin B nucleus.
Echinocandin cyclopeptides are natural antifungal products. Included in the Echinocandin cyclopeptide family are natural products such as Echinocandin B (ECB), Echinocandin C, Aculeacin Axcex3, Mulundocandin, Sporiofungin A, Pneumocandin A0, WF11899A, and Pneumocandin B0. These are typically produced by culturing various microorganisms. For example, Echinocandin B is produced from the fermentation of the fungus, Aspergillus Nidulans. 
In the search for more active materials, the natural products have been modified in a variety of ways. One of the most common has been replacement of the N-acyl side chain on the natural product to produce a semi-synthetic derivative. For example, U.S. Pat. Nos. 4,293,489; 4,320,052; 5,166,135; and 5,541,160; and EP 359529; 448353; 447186; 462531; and 561639 describe a variety of N-acyl derivatized Echinocandin compounds with varying degrees of antifungal activity.
The N-acyl derivatives are produced by deacylating the natural product followed by reacylation with a different acyl group. Deacylation is typically achieved by means of an enzyme (e.g., deacylase enzyme). The deacylase enzyme may be obtained from the microorganism Actinoplanes utahensis or Pseudomonas species (see i.e., U.S. Pat. Nos. 4,293,482 and 4,304,716; and EP 460,882). The deacylated compound is typically referred to as the nucleus of the corresponding natural product (e.g., the deacylated product of Echinocandin B is referred to as the Echinocandin B nucleus (ECBN)). Unfortunately, both the acylated and unacylated products are difficult to purify due to their limited solubility and amorphous state. In addition, the free amino compound (e.g., ECBN) is generally unstable and subject to ring opening.
It is well-known in the art that crystalline materials in general are easier to purify than their amorphous counterparts. Hence, it is desirable to produce cyclopeptide compounds in their crystalline state to obtain optimal purity. Since the potency of the final pharmaceutical product is dependent upon the purity of the intermediates used to make the final product, improvements in purity at any stage of the manufacturing process is highly desirable. Ideally, the contaminants are removed at the earliest stage possible in the manufacturing process. Hence, there is a need for a process that simplifies and improves the purification of cyclopeptide compounds containing a free amino group prior to subsequent attachment of an amino substituent.
The present invention provides a method for forming a crystalline echinocandin nucleus salt from its mixed broth or partially purified process streams by the steps of (i) concentrating a solution comprising an echinocandin nucleus or amorphous salt thereof, an aldehyde impurity and a solvent by means of a nanofiltration process to form a concentrate; (ii) adding an aldehyde derivatizing agent; (iii) adjusting the pH to a value less than 4.0 (preferably between about 2.0 and about 3.0); (iv) adding an acid or metal salt; and (v) cooling the concentrate to crystallize an echinocandin nucleus salt having an anion corresponding to the anion of the acid or metal salt added in step (iv). A seed crystal may optionally be added to initiate crystallization.
In another embodiment of the present invention, a process for exchanging the anion of an Echinocandin ammonium salt (including simple derivatives thereof) is provided as well as various forms of crystalline echinocandin nucleus salts.
xe2x80x9cEchinocandin compoundsxe2x80x9d refers to compounds having the following general structure including any simple derivatives thereof: 
wherein R is a hydrogen or xe2x80x94C(O)Rxe2x80x2 where Rxe2x80x2 is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or heteroaryl group; R1 is xe2x80x94H or xe2x80x94OH; R2 is xe2x80x94H, xe2x80x94NH2 or xe2x80x94CH3; R3 is xe2x80x94H, xe2x80x94CH3, xe2x80x94CH2CONH, or xe2x80x94CH2CH2NH2; R4 is xe2x80x94H or xe2x80x94OH; R5 is xe2x80x94OH, xe2x80x94OSO3H, or xe2x80x94OPO2HRa, where Ra is hydroxy, C1-C6 alkyl, C1-C6 alkoxy, phenyl, phenoxy, p-halophenyl, p-halophenoxy, p-nitrophenyl, p-nitrophenoxy, benzyl, benzyloxy, p-halobenzyl, p-halobenzyloxy, p-nitrobenzyl, or p-nitrobenzyloxy; R6 is xe2x80x94H, xe2x80x94OH, or xe2x80x94OSO3H; R7 is xe2x80x94H or xe2x80x94CH3; and pharmaceutically acceptable salts, esters, hydrates or solvates thereof. Also included within the meaning of echinocandin are the various enantomeric forms of structure I illustrated above even though specific chiral centers are depicted. xe2x80x9cEchinocandin nucleusxe2x80x9d refers to the deacylated Echinocandin compound where R is a hydrogen. xe2x80x9cECBNxe2x80x9d refers to the Echinocandin B nucleus where R1, R4 and R5 are hydroxyl groups, R2, R3, and R7 are methyl groups; and R and R6 are hydrogens.
xe2x80x9cAlkylxe2x80x9d refers to a hydrocarbon radical of the general formula CnH2n+1, containing from 1 to 30 carbon atoms unless otherwise indicated. The alkane radical may be straight (e.g. methyl, ethyl, propyl, butyl, etc.), branched (e.g., isopropyl, isobutyl, tertiary butyl, neopentyl, etc.), cyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, methylcyclopentyl, cyclohexyl, etc.), or multi-cyclic (e.g., bicyclo[2.2.1]heptane, spiro[2.2]pentane, etc.). The alkane radical may be substituted or unsubstituted. Similarly, the alkyl portion of an alkoxy group or alkanoate have the same definition as above.
xe2x80x9cAlkenylxe2x80x9d refers to an acyclic hydrocarbon containing at least one carbon carbon double bond. The alkene radical may be straight, branched, cyclic, or multi-cyclic. The alkene radical may be substituted or unsubstituted.
xe2x80x9cAlkynylxe2x80x9d refers to an acyclic hydrocarbon containing at least one carbon carbon triple bond. The alkyne radical may be straight, or branched. The alkyne radical may be substituted or unsubstituted.
xe2x80x9cArylxe2x80x9d refers to aromatic moieties having single (e.g., phenyl) or fused ring systems (e.g., napthalene, anthracene, phenanthrene, etc.). The aryl groups may be substituted or unsubstituted.
xe2x80x9cHeteroarylxe2x80x9d refers to aromatic moieties containing at least one heteroatom within the aromatic ring system (e.g., pyrrole, pyridine, indole, thiophene, furan, benzofuran, imidazole, pyrimidine, purine, benzimidazole, quinoline, etc.). The aromatic moiety may consist of a single or fused ring system. The heteroaryl groups may be substituted or unsubstituted.
Within the field of organic chemistry and particularly within the field of organic biochemistry, it is widely understood that significant substitution of compounds is tolerated or even useful. In the present invention, for example, the term alkyl group allows for substituents which is a classic alkyl, such as methyl, ethyl, propyl, hexyl, isooctyl, dodecyl, stearyl, etc. The term specifically envisions and allows for substitutions on alkyls which are common in the art, such as hydroxy, halogen, alkoxy, carbonyl, keto, ester, carbamato, etc., as well as including the unsubstituted alkyl moiety. However, the substituents should be selected so as to not adversely affect the pharmacological characteristics of the compound or adversely interfere with the use of the medicament. Suitable substituents for any of the groups defined above include alkyl, alkenyl, alkynyl, aryl, halo, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, mono- and di-alkyl amino, quaternary ammonium salts, aminoalkoxy, hydroxyalkylamino, aminoalkylthio, carbamyl, carbonyl, carboxy, glycolyl, glycyl, hydrazino, guanyl, and combinations thereof.
xe2x80x9cSolvatexe2x80x9d means an aggregate that comprises one or more molecules of the solute, such as Compound I, with one or more molecules of a solvent, such as water, ethanol, and the like.
xe2x80x9cSuitable solventxe2x80x9d refers to any solvent, or mixture of solvents, inert to the ongoing reaction that sufficiently solubilizes the reactants to afford a medium within which to effect the desired anion exchange or salt formation.
xe2x80x9cMixed brothxe2x80x9d refers to a conversion mixture where the fermentation broth is treated directly with a deacylating enzyme without purification to produce the deacylated product (e.g. ECBN).
Crude mixtures of cyclic peptides described herein may be prepared by fermentation of known microorganisms as described in the art. The subsequent deacylation is typically carried out enzymatically using a deacylase enzyme by known materials and procedures described in the art.
For example, the cyclic peptide I where R1 and R4 are each hydroxy, R2, R3 and R7 are each methyl (i.e., cyclic nucleus corresponding to A-30912A) may be prepared using the procedure detailed in U.S. Pat. No. 4,293,482. The cyclic peptide II(a) where R1 is hydroxy, R2, R3 and R7 are each methyl, and R4 is hydrogen (i.e., cyclic nucleus corresponding to A-30912B) may be prepared using the procedure detailed in U.S. Pat. No. 4,299,763. Aculeacin may be prepared using the procedure detailed in U.S. Pat. No. 3,978,210. The cyclic peptide I where R3 is CH2C(O)NH2, R7 is methyl, R2 is hydrogen, and R1 and R4 are hydroxy may be prepared using the procedure detailed in U.S. Pat. No. 5,198,421.
Fermentation and mixed broths contain a number of related by-products that are very difficult to separate from the desired cyclopeptide product. Reversed phase, liquid chromatography (RP-LC) has been used in the past with reasonable success; however, the need for higher purity compounds demands even more improved methods of purification.
Products isolated from a mixed broth solution or a fermentation process are generally prefiltered to remove particulates. Prefiltration may be accomplished by any number of means known in the art including gravity filtration, vacuum filtration through a ceramic filter which may or may not include a Celite(trademark) filter aid, etc. Solids in the fermentation broth may also be removed by centrifugation followed by decanting the liquid from the solids. Concentrates from a mixed broth refer to those acquired directly from the filtration or centrifugation of the fermentation mixed broth.
If the filtered solution requires further purification, the concentrated solution may be separated using preparative liquid chromatography prior to any crystallization attempts. Those concentrates that originate from chromatographic partitions serve as an example of solutions from a partially purified process stream and are referred to as a xe2x80x9cpolished concentrate.xe2x80x9d
Any chromatographic method well-known in the art may be used to provide the desired separation of products. Preferred chromatographic methods employ the use of reverse-phase media with an acidic elution scheme. Preferably, an eluent containing acetic acid. For example, the material may be purified using the chromatographic method described in Kroeff, et al. filed Dec. 9, 1998 entitled xe2x80x9cPurification of Echinocandin Cyclopeptide Compounds.xe2x80x9d The purification method includes adsorbing the mixture onto a hydrophobic, reversed phase chromatographic media and eluting with a continuous nearly linear acetic acid gradient ranging from 0.1% acetic acid to 10.0% acetic acid by volume in water, preferably from 0.5% (pH=5.5) to 4.0% (pH=2.5) acetic acid.
To crystallize the ECBN salt, the solution from the mixed broth or collected partitions from the chromatographic process are first concentrated. Conventionally, the solution was concentrated by means of an evaporative method (e.g., distillation). However, Applicants have discovered that a nano-filtration system provides a more efficient and higher quality concentrate. The process involves a 200 fold concentration of a dilute (approx. 1 g/liter) solution of the cyclopeptide nucleus on an approximately 400 molecular weight reverse osmosis membrane. The membrane retains the cyclopeptide nucleus while allowing lower molecular weight impurities to pass through. The nano-filtration method provides several advantages over the conventional evaporative methods such as, higher potency, eliminates the need for freeze drying the nucleus, shorter cycle time, and significant reduction of degradation products during concentration. Unlike distillation, nano-filtration allows one to produce a concentrate having a weight percent between about 18 and 22% without significant degradation.
In addition to other related impurities, the fermentation broth for Echinocandin B contains varying levels of a tripeptide-aldehyde (Asn-Gln-Leu-H) by-product having the following chemical structure (Ia). The tripeptide-aldehyde by-product under goes deacylation as well as Echinocandin B during the enzymatic deacylation process to form the corresponding deacylated tripeptide-aldehyde (Ib). 
where R is C(O)CH2CH(OH)C9H19 (Ia-fermentation by-product) or a hydrogen (Ib-deacylation by-product from a mixed broth).
Surprisingly, the retention time of the deacylated tripeptide-aldehyde is very similar to ECBN in reversed phase, liquid chromatography (RP-LC), even under optimum elution conditions, thus making it very difficult to separate the deacylated tripeptide-aldehyde (Ib) from the desired ECBN. The nano-filtration process also does not sufficiently remove the deacylated tripeptide-aldehyde. It has now been shown that the tripeptide impurity influences the ability to crystallize the ECBN salt. Although not wishing to be bound by any one theory, it is believed that the tripeptide impurity (Ib) forms a weak complex with the ECB Nucleus in solution which serves to decrease, or otherwise inhibit the rate of ECB Nucleus crystallization, thus contributing to poor product recovery. Consequently, the tripeptide by-product is preferably removed or modified prior to the isolation of crystalline ECBN.
The tripeptide-aldehyde by-product may be modified in the ECBN concentrate by reacting the aldehyde with a derivatizing agent prior to crystallization. The derivatizing agent selectively interacts with the aldehyde thus decreasing or eliminating any interaction between the aldehyde and the ECBN. xe2x80x9cDerivatizing agentxe2x80x9d refers to a reagent capable of interacting (i.e., reaction or complexation) with the aldehyde functionality of the tripeptide by-product to produce an intermediate that is sufficiently different in hydrophobicity to allow separation of the tripeptide intermediate from the desired ECBN salt. For example, the solubility of the aldehyde is increased such that the ECBN salt selectively crystallizes from solution leaving the aldehyde in solution. Suitable derivatizing agents include sodium bisulfite, hydrazine, hydroxyl amine and semicarbazide hydrochloride. At least one equivalent of derivatizing agent is added per equivalent of aldehyde impurity. Preferably, a slight excess of derivatizing agent is added (i.e., approximately 1.2 equivalents).
An organic or inorganic acid is added to the concentrate to adjust the pH of the concentrate solution to less than 4.0, preferably between about 4.0 and 2.0, more preferably between about 3.5 and about 2.5. The optimum pH (i.e., degree of protonation) will depend upon the local chemical environment of the amine function. In other words, the pH is adjusted such that formation of the ammonium salt is favored. The ECBN salt may be crystallized from the acidic concentrate by adding an acid or metal salt containing the desired anion followed by slowly cooling the mixture to initiate crystallization. The acid/metal salt may be added in portions. The portions may be added in equal or unequal amounts. Portion wise addition appears to control the crystal growth process. Typically, the first portion contains nearly twice the amount of the second or third portion. Preferably, the metal salt is added in portions at different temperatures. For example, the first portion of metal salt is added between about 22 and 28xc2x0 C., the second portion added between about 20 and 15xc2x0 C., and the third portion added between about 8 and 12xc2x0 C. Lowering the temperature from 28xc2x0 C. to about 10xc2x0 C. helps to decrease the solubility of the ECBN salt and thus assists in the crystallization of the ECBN salt; however, further lowering of the temperature below 10xc2x0 C. did not appear to significantly effect the solubility of the ECBN salt. The increased amount of acid/metal salt added to the concentrate is believed to not only provide a rich anion source, but also reduces the solubility of the ECBN salt. The total amount of acid/metal salt added to the concentrate is generally between about 14 and 16 weight percent of the concentrate. Preferably, a seed crystal is added to assist the initiation of the crystallization process.
When the cyclopeptide is the nucleus of echinocandin B, the acetate salt is an amorphous solid. Applicants have discovered that the anion of the amorphous ammonium cyclopeptide salt can be easily exchanged in the presence of an alternative anion source (an acid or metal salt) to form a crystalline salt. For example, the HPLC partitions containing the ECBN is typically in the form of an ammonium acetate salt since the eluent is acetic acid. The anion-exchange may be accomplished by adding the appropriate acid/metal salt which serves as the alternative anion source at any step prior to crystallization. For ECBN, a preferred anion source is HCl/sodium chloride.
In summary, the formation of an ECBN salt includes the steps of: (i) concentrating a solution containing ECBN or amorphous salt thereof and an aldehyde impurity using a nanofiltration process; (ii) adding a derivatizing agent (preferably sodium bisulfite) which interacts with the aldehyde impurity; (iii) adjusting the pH to less than 4.0; (iv) adding an acid or metal salt (preferably NaCl); and (v) cooling the mixture to initiate crystallization of the ECBN salt. A seed crystal of ECBN salt may optionally be added to help initiate crystallization. Preferably, the sodium chloride is added in three portions (the first portion is added between about 22 and 28xc2x0 C.; the second portion is added between about 15 and 20xc2x0 C.; and the third portion is added between about 8 and 12xc2x0 C.). In addition, the first portion, preferably, contains nearly twice the amount of sodium chloride by weight as the second or third portion.
The anion of an isolated ECBN salt may be exchanged by slurrying the cyclopeptide ammonium salt with an acid salt (or metal salt) containing the desired anion in a suitable solvent, heating the slurry to dissolve the reactants, and then cooling the solution to form the desired crystalline salt.
The crystalline forms offer several advantages such as easier isolation of the cyclopeptide from the mixed fermentation broth and/or process streams, improved purification of intermediates, improved shelf-life, and increased yields of the final acylated product. The degree to which each of these advantages are realized may be dependent upon the particular salt form and the process by which the salt is produced.
The crystalline salt may be isolated in a variety of crystalline forms (e.g., simple salt and inner-salt forms, solvated and/or hydrated forms, etc.). A simple protonated ammonium salt may be in the form of a mono- or di-acid addition salt, such as CPxe2x80x94NH3+Axe2x88x92, (CPxe2x80x94NH3+)2Axe2x88x922, and (CPxe2x80x94NH3+M+)Axe2x88x922 where CPxe2x80x94NH3+ represents the cyclopeptide containing a protonated primary amino group (e.g., ECBN), A is a mono- or di-valent anion and M+ is a mono-valent metal. Suitable monovalent anions include chloride, bromide, iodide, dihydrogen phosphate, hydrogen sulfate, hydrogen oxalate, hydrogen tartrate, benzoate, methanesulfonate and p-toluenesulfonate. Suitable divalent anions include sulfate, oxalate, hydrogen phosphate, tartrate and fumarate. Suitable metal cations include ammonium, lithium, sodium, potassium and tetraalkylammonium.
Inner-salt forms may be represented by formulae such as (CPxe2x80x94NH3+Axe2x88x92)(M+Axe2x88x92) and ((CPxe2x80x94NH3xe2x88x92)2Axe2x88x922)(M+2Axe2x88x922), where M+2 is a divalent metal. Suitable divalent metals include calcium and magnesium.
In addition to the basic salt forms discussed above, the salt can be isolated as a solvate. Examples of solvated forms include those with the following chemical formulae: (CPxe2x80x94NH3+Axe2x88x92)(H2O)a(S)b where S is an organic solvent and the subscripts a and b represent solvate stoichiometry. Suitable solvate solvents include methanol, ethanol, ethylacetate, acetone, acetonitrile, tetrahydrofuran and toluene.
The non-solvated and solvated forms may exhibit polymorphism. For example, the crystalline form may be dependent upon the conditions for crystallization. Even though the stoichiometry may be the same, there may exist different three dimensional solid phase crystalline structures with different physical and chemical properties.
It will be understood by those skilled in the art that the following serves as illustrative examples and that other cyclopeptide ammonium salts can be purified or produced using the procedures described below. All references cited herein are hereby incorporated herein by reference.